Serotonin (5-Hydroxytryptamine, 5-HT) is a well characterized neurotransmitter and vasoactive amine which has been implicated in common disorders involving central nervous, gastrointestinal, cardiovascular and pulmonary systems (for review see Berger, M. et al., Annu Rev Med. (2009), 60, 355-366). Peripheral 5-HT is mainly synthesized and released by the enterochromaffin cells in the gut. When reaching the blood stream it is sequestered inside platelets. Under normal conditions the level of free 5-HT in plasma is low and strictly regulated by specific 5-HT transporters present on the surface of e.g. platelets as well as by 5-HT degrading enzymes. Upon activation platelets release 5-HT and a local increase in 5-HT concentration is observed. Over the years evidence has gathered that 5-HT has a significant role in the functioning of the mammalian body. For example it has been shown to regulate processes like cardiovascular function, bowel motility and bladder control. 5-HT and the 5-HT receptor system have also been associated with the modulation of pain and more specifically, the 5-HT2 receptors have been shown to play an important role in the inflammatory pain process (Cervantes-Duran C. et al., Neuroscience (2012) 232, 169). Furthermore, several studies have shown that the 5-HT system has an important role in the regulation of inflammation. The exact function of 5-HT on inflammation is poorly recognized though, with only a few and inconsistent published reports.
A greater understanding of 5-HT function has emerged with the characterization of its, at least, 14 different human receptors which are grouped into subfamilies based on their structural and pharmacological differences. Each receptor exhibit unique distribution and shows various preference for different ligands. The receptors are all G protein-coupled receptors, with the exception of the 5-HT3 receptor, which is a ligand-gated ion channel. Several of the 5-HT receptors have been linked to 5-HT's effects in inflammation.
The 5-HT2 receptor family consists of 3 subtypes, 5-HT2A, 5-HT2B and 5-HT2C. The 5-HT2 receptors share significant sequence homology at the amino acid level and couple to the Gq family of the G proteins. An important role of 5-HT2 receptors in inflammation has been shown in the kaolin/carrageenan-induced rat arthritis model in which the 5-HT2A receptor antagonist ketanserin suppressed oedema formation and hyperalgesia. Also, mianserin, a 5-HT2A/2C receptor antagonist, has been shown to inhibit cytokine production in human RA synovial membrane cultures.
The link between the 5-HT2B receptor and inflammation is less investigated, however, the involvement of 5-HT2B receptor signalling in 5-HT-induced production of IL-1β, IL-6 and TNF-α in mouse cardiac fibroblasts has been shown (Jaffre F. et al., Circulation (2004) 110(8), 969-74). The mRNA expression of the 5-HT2B receptor on inflammatory cells such as macrophages and fibroblasts makes it important to investigate 5-HT2B as target for modulating an inflammatory response in diseases such as rheumatoid arthritis. The use of N-benzylidene aminoguanidines as 5-HT2B receptor antagonist for this purpose has been described in WO2011012868A1.
The 5-HT2B receptor has previously been linked to pulmonary arterial hypertension (PAH) (Thomas M. et al., Pharmacol Ther. (2013) 138(3), 409-17) and the phenotype of the 5-HT2B receptor knock-out mice shows its importance for heart development. It demonstrates that 5-HT via the 5-HT2B receptor regulates differentiation and proliferation of developing and adult heart. Furthermore, over-expression of the 5-HT2B receptor in mice leads to cardiac hypertrophy (Nebigil C. G. et al., Circulation. (2003) 107(25), 3223-9).
In agreement with this, 5-HT and its receptors, 5-HT2A and 5-HT2B in particular, have been implicated in the etiology of several fibrotic disorders including retroperitoneal fibrosis, carcinoid heart disease, systemic sclerosis, liver and lung fibrosis. Fibrosis is actually a feature of many different types of chronic respiratory diseases including IPF, PAH, COPD and asthma. A mechanistic link between fibrosis and 5-HT was first reported in the 1960s for a condition called carcinoid syndrome which is caused by neuroendocrine carcinoid tumours that secrete vast quantities of 5-HT. The syndrome is characterized by tissue fibrosis that particularly affects cardiac valves but also impacts on other organs including lung and skin. Subsequently, agonism on the 5-HT2B receptor has been implicated in fibrosis caused by fenfluramine used in the treatment of obesity and psychiatric disorders. Fibrosis is characterized by enhanced fibroblast/myofibroblast proliferation and activation which results in an altered extracellular matrix deposition which ultimately results in organ failure (Mann, D. A. and Oakley F., Biochim Biophys Acta. (2013) 1832(7), 905-10).
An important mediator of the fibrotic process is transforming growth factor beta, TGF-β. This cytokine modulates a variety of physiological processes through transcriptional regulation. In human lung fibroblasts, TGF-β is well-known for inducing myofibroblast differentiation with increased levels of alpha-SMA in intracellular stress fibers as well as an increased matrix deposition. A lot of evidence support a role of 5-HT in fibrosis although the exact mechanism how 5-HT promotes fibrosis is not defined. 5-HT has been shown to increase the mRNA levels of TGF-β via the 5-HT2B receptor and in models of systemic sclerosis human dermal fibroblasts have a dose-dependent increase of TGF-β mRNA in response to 5-HT as well as an increased expression of the 5-HT2B receptor. This results in an increased mRNA expression of collagen 1a1, collagen 1a2 and fibronectin. The effects of 5-HT on matrix synthesis were blocked by a 5-HT2B receptor antagonist or by transfected 5-HT2B siRNAs. The same study showed that selective 5-HT2B receptor antagonists prevent bleomycin-induced dermal fibrosis in vivo (Dees C., et al., J. Exp. Med. (2011) 208(5), 961-72). In other fibrotic diseases such as liver fibrosis, treatment with 5-HT2B receptor antagonists resulted in attenuated fibrogenesis in an in vivo model of chronic liver disease (Ebrahimkhani, M. R., et al., Nat Med. (2011) 17(12), 1668-73). Further support for 5-HT and fibrosis is found in patients suffering from IPF that have an increased expression of 5-HT2A and 5-HT2B receptors in the fibrotic lung. Another study identified strong fibroblast expression of 5-HT2B receptor in fibroblastic foci in human lung samples from IPF patients. In addition, treatment with Terguride, a 5-HT2A and 5-HT2B receptor antagonist, reduces the expression of type I collagen in TGF-β1 stimulated human lung fibroblasts. This anti-fibrotic effect is also seen after treatment with 5-HT2A and 5-HT2B receptor antagonists in the bleomycin (BLM)-induced lung fibrosis model in mice (Konigshoff M., et al. Thorax. (2010); 65(11), 949-55 and Fabre A., et al. Eur Respir J. (2008) 32(2), 426-36).
5-HT2B Antagonists
Many 5-HT2B antagonists of variable structural classes have been described in the literature, such as in WO2011012868A1. Two recent reviews on the subject enlist such compounds, their intended uses, and their state of development (Poissonnet, G., et al., Mini-Reviews in Medicinal Chemistry (2004), 4(3), 325-330 and Brea, J. et al., Current Topics in Medicinal Chemistry (Sharjah, United Arab Emirates) (2010), 10(5), 493-503). These include the di-ureas SB206553 and SB215505, the piperazine derivative EGIS-7625, the 2-amino-4-naphthyl-pyrimidine MT-500 (RS127445), thioxanthene structures, the ergot derivative terguride, tetrahydro-β-carbolines, the thienopyrimidine PRX-08066, and quinoline derivatives. More recent examples of 5-HT2B antagonists that also contain a guanidine moiety, are disclosed in US2009062363A1.
Structurally related 5-HT2B antagonists are N-benzylidene amminoguanidines, such as N-(2-chloro-3,4-dimethoxybenzylidene-amino)guanidine (WO2011012868A1).
1-Amidino-3-Aryl-2-Pyrazolines
The compound class 1-amidino-3-aryl-2-pyrazolines was first reported in the 1950s describing synthetic preparative methods in which aryl Mannich bases were condensed with aminoguanidine (Scheme 1),
see: Scott, F. L. and Reilly, J., Chemistry & Industry (London, United Kingdom) (1952), 907-8 and Nitrogen systems. XIV. Scott, F. L. and Scott, M. T., Chimia (1958), 12, 148-50. Some derivatives, typically substituted at the 4- or 5-position in the pyrazoline ring and/or at the amidine group, have later been developed into pharmacologically active compounds. These include 4-aryl-N-sulfonamides as CB1-antagonists and potassium channel modulators (WO2001070700 A1 and WO2007125049A1), 4-heterocyclyl derivatives as PAR-1 antagonists (WO2005007157A1), N-sulfonamides as 5-HT6 antagonists (WO2008034863A2), and 5-aryl derivatives as necroptosis inhibitors (Jagtap, P. G., et al., J. Med. Chem. (2007), 50(8), 1886-1895), as MAO-inhibitors (Sahoo, A. et al., Bioorganic & Medicinal Chemistry Letters (2010), 20(1), 132-136 and Jagrat, M. et al., Bioorganic & Medicinal Chemistry Letters (2011), 21(14), 4296-4300), and as antimicrobials (Ferreras, J. A. et al., Bioorganic & Medicinal Chemistry Letters (2011), 21(21), 6533-6537).
Also, the para-substituted 1-amidino-3-aryl-4-methyl-2-pyrazolines below have been reported as anti-inflammatory and analgesic agents, however, with no mode of action described. (Abd-El G., et al. Arch Pharm Res (2012), 35(5), 807-821).
